Saadullah Khan  ( Department of Biotechnology and Genetic Engineering, Kohat University of Science and Technology, Kohat, KPK, Pakistan. )
Anwar Kamal Khan  ( Department of Biotechnology and Genetic Engineering, Kohat University of Science and Technology, Kohat, Khyber Pakhtunkhwa, Pakistan; )
Malaika Hamid  ( Department of Biotechnology and Genetic Engineering, Kohat University of Science and Technology, Kohat, Khyber Pakhtunkhwa, Pakistan; )
Muhammad Nazif  ( Department of Biotechnology and Genetic Engineering, Kohat University of Science and Technology, Kohat, Khyber Pakhtunkhwa, Pakistan; )
Muhammad Abbas  ( 3Department of Prosthodontics, Ziauddin College of Dentistry, Karachi. )
Sher Alam Khan  ( Department of Biotechnology and Genetic Engineering, Kohat University of Science and Technology, Kohat, Khyber Pakhtunkhwa, Pakistan; )
Bushra Khan  ( Department of Biochemistry, Abdul Wali Khan University, Mardan, Khyber Pakhtunkhwa, Pakistan; )
Muzammil Ahmad Khan  ( Gomal Centre of Biochemistry and Biotechnology, Gomal University, D.I. Khan, Khyber Pakhtunkhwa, Pakistan )
Abid Jan  ( Department of Biotechnology and Genetic Engineering, Kohat University of Science and Technology, Kohat, Khyber Pakhtunkhwa, Pakistan; )
Baharullah Khattak  ( Department of Microbiology, Kohat University of Science and Technology, Kohat, Pakistan )
Waheed Ullah  ( Department of Microbiology, Kohat University of Science and Technology, Kohat, Pakistan. )
Noor Muhammad  ( Department of Biotechnology and Genetic Engineering, Kohat University of Science and Technology (KUST), Kohat, Khyber Pakhtunkhwa, Pakistan )

Abstract

Primitive epidermis develops the nail apparatus. Nails have a strong and inflexible nail plate at the end of each digit. Very few genes responsible for causing nonsyndromic form of nail dysplasia have been reported. In the current study, peripheral blood samples were collectedfrom three unaffected individuals and four affectedindividuals of Family A, while blood from two affected and three unaffected individuals were taken of Family B. Genotyping in both the families was performed using highly polymorphic short tandem repeat microsatellite markers. Sanger sequence of the FZD6 gene was performed and analysed for segregation analysis. A comparative modelling approach was used to predict the three-dimensional structures of FZD-6 protein using Modeller 4. Linkage analysis mapped a disease locus on chromosome 8q22.3, harbouring FZD6. Targeted Sanger sequencing of all the coding exons of FZD6 revealed a nonsense sequence variant in pedigree A, whereas a missense sequence variant in pedigree B. Finding and literature indicates the disease spectrum of Pakistani population with claw-shaped nail dysplasia, particularly in families of Pashtun origin.

Keywords:Non-syndromic nail dysplasia, Chromosome 8q22.3, FZD6, Missense and nonsense sequence variants.https://doi.org/10.5455/JPMA.9200

Introduction

At the 9th week of gestation, initiation of the development of the human nail occurs and is completed during the 5thmonth of pregnancy, with the development of toenails lagging approximately a month behind the fingernails.1Matrix produces the nail, and its growth occurs over the nail-bed. The mature nail plate grows continuously throughout life as a result of matrix epithelial cell differentiation and consists of a number of hard and soft keratin molecules embedded in an amorphous matrix.1,2Most inherited nail disorders manifest either with nail hypoplasia or nail hypertrophy.3Non-syndromic congenital nail disorder (NDNC) may beinherited in either an autosomal-dominant or autosomalrecessivefashion. It has been classified into 10 different types (NDNC1-10), which are described in Online Mendelian Inheritance in Man (OMIM).4 NDNC1 and NDNC2 are termed 20-nail dystrophy and koilonychias, respectively,5,6 and the genes for either condition remain unknown. NDNC3, or leuconychia, has genetic address to chromosome 3p21.3-p22 with pathogenic variants reported in the PLCD1 gene.7Complete absence of nail (NDNC4) was reported to result from sequence variants in the RSPO4.8 Hereditary distal onycholysis and partial absence of nails were termed NDNC5 and NDNC6, respectively.9,10NDNC7, characterised by longitudinal streaks in nails, nail plate thinning, and poorly developed lunula, was mapped at chromosome 17p13.11,12The genes for NDNC5-7 have not been identified yet. Sequence variants in the COL7A1 gene were reported for NDNC8 which consists of pure toenail dystrophy with autosomal dominant inheritance.13Onychodystrophy (NDNC9) was previously mapped to chromosome 17q25.1-17q25.3 for which the gene remains to be identified.14Thick, shiny, hard and pigmented nails (NDNC10) are due to sequence variants in FZD 6on chromosome 8q 22.3.15The current study was planned to see the association of sequence variants in FZD6 with NDNC10 in Pashtun families.

Materials and Methods

The study was conducted from March to September 2017 after approval from the institutional review board of Kohat University of Science and Technology (KUST ), Kohat, Khyber Pakhtunkhwa (KP), Pakistan. Informed written consent for clinical and molecular investigation was

obtained from all the subjects, and the study wasconducted in accordance with the Declaration of Helsinki Principles.16 Two families with nail dystrophy were recruited from Pashto-speaking region of the KP province (Figure 1A-B). No evidence of any other abnormality of ectodermal appendages, including sweat glands, hair and teeth, was noted in any of the affected individuals in both families. The affected siblings in both families were born to healthy parents. Clinical examinations were performed at the respective local government hospitals where the families resided. Peripheral blood samples were collected from four affected (IV-2, IV-3, IV-4, IV-6) and three unaffected individuals (III-1, III-2, IV-1) of Family A, while blood from two affected (IV-1, IV-2) and three unaffected individuals (III-1, III-2, IV- 3) were taken from Family B. From the blood lymphocytes, genomic deoxyribonucleic acid (DNA) was extracted using phenol-chloroform method17and commercially available kit Nucleospin® Blood (Macherey-Nagel, Germany). Genotyping in both the families was performed using

highly polymorphic shor t tandem repeat (STR) microsatellite markers, with an average heterozygosity of 0.70. The polumerase chain reaction (PCR) products were resolved in 8.0% non-denaturing polyacrylamide gel, and genotypes were assigned by visual inspection. Haplotype analysis of the genotyped markers revealed a region of homozygosity among affected individuals of both the families on chromosome 8q22.3. FZD6, a cause of NDNC10, lies in this region, and thus appeared to be a strong candidate. Primers for PCR-amplification of FZD6 were designed using Primer3 software.18Sanger sequencing was performed after standard purification, using a commercially available BigDye Terminator v3.1Cycle Sequencing Kit (Life Technologies, Carlsbad, CA,USA). Segregation analysis was carried out using the BioEdit Sequence Alignment Editor version 7.0.5.3.19Pathological characteristics of the sequence variant were analysed using PolyPhen-2 algorithm.20Sequences of the primers were as follows:

A comparative modelling approach was used to predict the three-dimensional (3D) structures of FZD-6 protein using Modeller 4.21 We used the X-ray crystal structure of the cysteine-rich domain of human FZD4 (Protein database [PDB] identity [ID]: 5BPB, sequence identity 39%) and human srGAP2 motif (PDB ID: 5I7D, sequence identity 36%), as a scaffold for the homology modelling of wild and mutant types of FZD-6 protein.22,23The models were visualized using Pymol software (https://pymol.org/).24

Results

The age of the affected members ranged 10-30 years at the time of the study. All the affected members showed severe form of nail dysplasia. The affected individuals of both pedigrees displayed features of shiny, hyperpigmented and hyperplastic nails. Few individuals had claw-shaped nails. Some of the affected individuals had hyponychia and onycholysis of both fingernails and toenails (Figure 1-C). The paternal and maternal family history in both the families did not reveal evidence for any type of nail ornail-associated dysplasia. Heterozygous carrier individuals had normal finger nails and were clinically indistinguishable from genotypically normal individuals. None of the affected members had any other associated abnormality of the teeth, hair, face and sweat glands. Genotyping data and haplotype analysis showed a single homozygous stretch on chromosome 8q22.3, present only in affec ted individuals of both pedigrees. Subsequently, all the exons and exon-intron boundaries of FZD6 were sequenced in one affected and parents of both the families. Upon identifying the sequence variants, the same exon of the gene was sequenced in rest of the affected and unaffected individuals of the respective family. Sequencing of the FZD6 revealed a single homozygous nonsense variant, changing glutamic acid to stop (c.1750G>T; Glu584*) in all the affected individuals of Family A that resulted in truncation. Sequencing analysis identified a missense variant, substituting glycine with aspartic acid (c.1266G >A; p.Gly422Asp) in all the affected individuals of Family B. Segregation analysis of FZD6 variants in both families was consistent with autosomal recessive inheritance. The sequence variants identified in Family A were: c.1750G>T; Glu584*. The sequence variants identified in Family B were: c.1266G >A; p.Gly422Asp. Residue Gly422 is located in the helix region which does not involve any binding with nearby residues (Figure 2D). Among the nearby residues, the Ser421 made two hydrogen bonding with Ile417 and Trp300, while the other residues, Leu419, Leu423 and Leu663, had interactions that were hydrophobic. The substitution of glycine by glutamic acid at position 422 established a local conformation change in the nearby residues interactionsas shown by the difference in bond lengths. In case ofE584* sequence variant, the protein was truncated dueto the premature stop codon and the loss subdomain washighlighted by magenta colour (Figure 2).

Discussion

In the present study two pedigrees collected from Khyber Pakhtunkhwa province were investigated. Affected individuals in both the families were showing pure autosomal recessive nail dysplasia phenotypes (NDNC10). The clinical features of all patients from these two families were in line with literature.15,25,26According to Human Gene Mutation Database (HGMD) Professional 2018.1,27there are 16 different sequence variants in the FZD6 reported as a cause of nail dystrophy, neural tubes defects and isolated nail dysplasia from different ethnicities around the world. The sequence variants (c.1750G>T; Glu584*)identified in Family A and (c.1266G >A; p.Gly422Asp) in Family B, have been described earlier.15,25,27,28Also, claw  development in mice has shown a regulatory role for FZD6-mediated Wnt signalling in the differentiation process of claw/nail formation.24The extensive prevalence of the variant (c.1750G>T; Glu584*) as reported in separate studies15,29points towards a founder effect and implies that the variation is due to a single alteration event on an ancestral chromosome 8.

Conclusion

The findings confirm previously described sequencevariants in FZD6 and indicates the disease spectrum of Pakistani population with claw-shaped nail dysplasia, particularly in families of Pashtun origin.

Disclaimer:None.

Conflict of interest:None.

Source of Funding:Financially the work presented here was supported through NRPU project (No.4857/ NRPU/ R&D/HEC 2014) by the Higher Education Commission (HEC), Islamabad, Pakistan.

References

1. Baran R, Dawber R, de Berker D, Haneke E, Tosti A. Baran and Dawber's diseases of the nails and their management. John Wiley & Sons; 2012.
2. Baran R, Bristow I, Dawber RP, Haneke E, Tosti A, editors. A text atlas of nail disorders: techniques in investigation and diagnosis. CRC Press; 2003.

3. Sprecher E. Genetic hair and nail disorders. Clin Dermatol. 2005;23:47-55.

4. Khan S, Basit S, Habib R, Kamal A, Muhammad N, Ahmad W. Genetics of human isolated hereditary nail disorders. Br J Dermatol. 2015;173:922-9.

5. Sehgal VN. Twenty nail dystrophy trachyonychia: an overview. J Dermatol. 2007;34:361-6.
6. Hellier FF. Hereditary koilonychia. Br J Dermatol Syph. 1950;62:213-5.

7. Kiuru M, Kurban M, Itoh M, Petukhova L, Shimomura Y, Wajid M, et al. Hereditary leukonychia, or porcelain nails, resulting from mutations in PLCD1. Am J Hum Genet. 2011;88:839-44.

8. Blaydon DC, Ishii Y, O'Toole EA, Unsworth HC, Teh MT, Ruschendorf F, et al. The gene encoding R-spondin 4 (RSPO4), a secreted protein implicated in Wnt signaling, is mutated in inherited anonychia. Nat Genet. 2006;38:1245-7.

9. Schulze HD. [Hereditary partial onycholysis with scleronychia]. Dermatol Wochenschr. 1966;152:766-75.
10. Charteris F. A case of partial hereditary anonychia. Glasgow Med J. 1918:207-

11. Hamm H, Karl S, Brocker EB. Isolated congenital nail dysplasia: anew autosomal dominant condition. Arch Dermatol. 2000;136:1239- 43.
12. Krebsova A, Hamm H, Karl S, Reis A, Hennies HC. Assignment of the gene for a new hereditary nail disorder, isolated congenital nail dysplasia, to chromosome 17p13. J Invest Dermatol. 2000;115:664-7.

13. Hammami-Hauasli N, Raghunath M, Kuster W, Bruckner-Tuderman L. Transient bullous dermolysis of the newborn associated with compound heterozygosity for recessive and dominant COL7A1 mutations. J Invest Dermatol. 1998;111:1214-9.

14. Rafiq MA, Ansar M, Pham T, Amin-ud-Din M, Anwar M, Haque S, et al. Localization of a novel locus for hereditary nail dysplasia to chromosome 17q25.1-17q25.3. Clin Genet. 2004;66:73-8.

15. Frojmark AS, Schuster J, Sobol M, Entesarian M, Kilander MBC, Gabrikova D, et al. Mutations in Frizzled 6 cause isolated autosomalrecessive nail dysplasia. Am J Hum Genet. 2011;88:852-60.

16. World Medical A. World Medical Association Declaration of Helsinki: ethical principles for medical research involving human subjects. JAMA. 2013;310:2191-4.

17. Sambrook J, Fritsch EG, Maniatis T. Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press: New York;1989.

18. Untergasser A, Cutcutache I, Koressaar T, Ye J, Faircloth BC, Remm M, et al. Primer3 - new capabilities and interfaces. Nucleic Acids Res. 2012;40:e115.

19. Hall TA. BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symposium Series. 1999: 95-8.

20. Adzhubei IA, Schmidt S, Peshkin L, Ramensky VE, Gerasimova A, Bork P, et al. A method and server for predicting damaging missensemutations. Nat Methods. 2010;7:248-9.
21. Webb B, Sali A. Protein Structure Modeling with MODELLER.Methods Mol Biol. 2017;1654:39-54.

22. Chang TH, Hsieh FL, Zebisch M, Harlos K, Elegheert J, Jones EY. Structure and functional properties of Norrin mimic Wnt for signalling with Frizzled4, Lrp5/6, and proteoglycan. Elife. 2015;4:e06554.

23. Sporny M, Guez-Haddad J, Kreusch A, Shakartzi S, Neznansky A, Cross A, Isupov MN, Qualmann B, Kessels MM, Opatowsky Y. Structural history of human SRGAP2 proteins. Mol biol evol. 2017;34:1463-78.

24. Seeliger D, de Groot BL. Ligand docking and binding site analysis with PyMOL and Autodock/Vina. J Comput Aided Mol Des. 2010; 24:417-22.

25. Raza SI, Muhammad N, Khan S, Ahmad W. A novel missense mutation in the gene FZD6 underlies autosomal recessive nail dysplasia. Br J Dermatol. 2013;168:422-5.

26. Naz G, Pasternack SM, Perrin C, Mattheisen M, Refke M, Khan S, et al. FZD6 encoding the Wnt receptor frizzled 6 is mutated in autosomal-recessive nail dysplasia. Br J Dermatol. 2012;166:1088-94.

27. Stenson PD, Mort M, Ball EV, Evans K, Hayden M, Heywood S, et al. The Human Gene Mutation Database: towards a comprehensive repository of inherited mutation data for medical research, genetic diagnosis and next-generation sequencing studies. Hum Genet. 2017;136:665-77.

28. Qasim I, Ahmad B, Khan MA, Khan N, Muhammad N, Basit S,et al. Pakistan Genetic Mutation Database (PGMD); A centralized Pakistani mutome data source. Eur J Med Genet. 2018;61:204-8.

29. Cui CY, Klar J, Georgii-Heming P, Frojmark AS, Baig SM, Schlessinger D, et al. Frizzled6 deficiency disrupts the differentiation process of nail development. J Invest D ermatol. 2013;133:1990-7.